The focus of this proposal is the study of oxidative stress in vivo using microdialysis sampling. Oxidative stress produces reactive oxygen species and ultimately free radicals that cause damage to DNA and other biomolecules. We propose to study the physiology of oxidative stress using in vivo microdialysis sampling at specific tissue sites during ischemia and reperfusion in the heart and brain. Microdialysis will be used to monitor oxygen free radicals (OFRs), an endogenous antioxidant that may serve as a protective agent (glutathione), and biomarkers for oxidative DNA damage (8-hydroxydeoxyguanosine) and lipid peroxidation (malondialdehye). In order to carry out the proposed studies, it will be necessary to first develop a suite of analytical methods to determine each of the target analytes in microdialysis samples. Microdialysis sampling results in samples of a few microliters or less. Therefore, methods based on capillary electrophoresis (CE), micellar electrokinetic chromatography (MEKC), and capillary electrokinetic chromatography (CEC) will be developed. The modified nucleosides and antioxidants are charged and electroactive. OFRs will be stabilized using radical trapping agents introduced through the microdialysis probe that produce electroactive products. All of these analytes will be detected using electrochemical methods. Malondialdehyde will be derivatized to a fluorescent analyte and determined by laser induced fluorescence. The role of OFR production will then be studied in vivo during ischemia and reperfusion in the heart and brain using microdialysis sampling and the newly developed methods of analysis. Studies will be aimed at elucidating the role of various proposed mechanisms of OFR formation during ischemia and reperfusion. In particular, the role xanthine oxidase plays in the production of OFRs in the brain will be investigated. OFR formation as a function of catecholamine release will be studied in both the heart and brain. Microdialysis will be used to locally administer inhibitors of xanthine oxidase and of catecholamine release and uptake in vivo to determine the role of these pathways.